A subset of non-small cell lung cancer (NSCLC) is driven by activating mutations of the epidermal growth factor receptor (EGFR). Mutant EGFR-driven lung cancers respond transiently to targeted therapy with the EGFR kinase inhibitors gefitinib and erlotinib, although the clinical efficacy of these inhibitors is limited by the development of drug resistance. The gatekeeper mutation EGFR T790M is the most prevalent resistance mechanism observed in the clinic, accounting for nearly 60% of drug resistance cases. Here, we propose to assess T790M predisposition and biology, and identify resistance mechanisms that emerge when T790M is targeted with a new class of mutant-selective EGFR inhibitors. The first objective is to study why some patients and cell lines develop EGFR T790M, while others become drug resistant through alternative mechanisms. It is not currently known whether EGFR T790M develops de novo in response to drug treatment, or if it is present at very low levels in treatment-naive tumors and becomes detectable due to a selective advantage conferred by drug treatment. To test these possibilities, treatment-nave cell lines and patient primary cells known to reproducibly become resistant through T790M will be subcloned and analyzed for T790M resistance before and after drug treatment. Characterizing resistance development in clonal subpopulations of cells will provide insight into the repertoire of resistanc mechanisms within a cell line, and help elucidate why T790M is the dominant resistance mechanism in some cellular contexts. Next, CRISPR-Cas9 genome editing will be used to engineer the T790M mutation into treatment-naive cell lines that become resistant through T790M or alternative resistance mechanisms. This will permit study of the T790M mutation in both endogenous and nonendogenous contexts, separating T790M development from any potential passenger mutations that arise during in vitro or ex vivo generation of drug resistance. The consequences of hindering T790M development or reverting EGFR T790M to the wild type codon (T790) will also be explored. The final aim of the proposed research is to characterize novel mechanisms of resistance to mutant-selective inhibitors that specifically target EGFR T790M, along with activating mutant EGFR. The efficacy of these inhibitors is being investigated in ongoing clinical trials. Modeling resistance to mutant-selective inhibitors using cell lines and patient samples will help predict resistance mechanisms that will emerge in the clinic. Taken together, the proposed experiments will provide insight into EGFR T790M development and treatment. These findings will have immediate implications for our understanding of resistance to targeted therapies, and could guide clinical decision-making, and ultimately prevent the development of drug resistance in patients with EGFR-mutant NSCLC.